A series of novel 2-oxo-quinoline derivatives containing α-aminophosphonates were designed and synthesized as antitumor agents. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay results demonstrated that some compounds exhibited moderate to high inhibitory activity against HepG2, SK-OV-3 and NCI-H460 tumor cell lines, and most compounds showed much lower cytotoxicity against HL-7702 normal cells than 5-FU and cisplatin. The action mechanism of representative compound <b>5b</b> was investigated by fluorescence staining assay, flow cytometric analysis and western blot (WB) assay, which indicated that this compound induced apoptosis and G<sub>2</sub>/M phase arrest accompanied by an increase in the production of intracellular Ca<sup>2+</sup> and reactive oxygen species (ROS) and affecting associated enzymes and genes.